Mucosal Cell Proliferation of the Rectal Stump in Ulcerative Colitis Patients After Ileorectal Anastomosis Francesco Tonelli, M.D., Franca Bianchini, Ph.D., Maura Lodovici, Ph.D., Rosa Valanzano, M.D., Giovanna Caderni, Ph.D., Piero Dolara, M.D. From the Departments of Clinical Physiopathology and Pharmacology, University of Florence, Florence, Italy Tonelli F, Bianchini F, Lodovici M, Valanzano R, Caderni G, Dolara P. Mucosal cell proliferation of the rectal stump in ulcerative colitis patients after ileorectal anastomosis. Dis Colon Rectum 1991;34:385-390. The proliferative activity and polyamine levels of the rectal epithelium in unoperated ulcerative colitis patients and in ulcerative colitis patients after total colectomy and ileorectal anastomosis were determined and compared with control subjects. Cell proliferation was evaluated in rectal biopsies by in vitro 3H thymidine incorporation by measuring the labeling index and the position of labeled cells along the crypt; polyamines were determined with a chromatographic method. In ulcerative colitis patients the labeling index was significantly increased, and labeled cells were shifted toward the upper part of the crypt when compared with controls. Ileorectal anastomosis patients showed a normalization of the labeling index and a distribution of labeled cells similar to controis. Polyamine levels were also increased in ulcerative colitis patients; in ileorectal anastomosis patients, the level of polyamines was decreased in respect to unoperated patients and return to normal values except for spermine. Because the increased proliferation and higher polyamine levels are related to increased colon cancer risk, our results confirm that ulcerative colitis is a risk factor for the development of carcinoma. Ileorectal anastomosis may reduce this risk through a normalization of mucosal cell proliferative activity and of some polyamine levels. [Key words: Cell proliferation; Ulcerative colitis; Ileorectal anastomosis]
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atients affected b y ulcerative colitis requiring c o l e c t o m y can p r e s e r v e c o n t i n e n c e and avoid a stoma b y two surgical p r o c e d u r e s : the ileorectal a n a s t o m o s i s (IRA) or the ileoanal anastomosis. T h e f o r m e r is a s i m p l e r t e c h n i q u e and, if p e r f o r m e d on p r o p e r l y s e l e c t e d patients, g u a r a n t e e s satisfactory functional results. ~' 2 H o w e v e r , leaving the d i s e a s e d r e c t u m in place m a y e x p o s e the patient to the risk
This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro, from CNR "Progetto Finalizzato FATMA"and by MPI, Italy. Address reprint requests to Professor Tonelli: Department of Clinical Physiopathology, Viale Morgagni 85, 50134 Florence, Italy. 385
of rectal carcinoma. This c o m p l i c a t i o n s e e m s to b e rare (varying f r o m 0 to 6 p e r c e n t in all patients3-5), but a reliable m e a s u r e of the risk is difficult to obtain b e c a u s e the risk of d e v e l o p i n g c a r c i n o m a increases with the duration of the disease, similar to what h a p p e n s in patients with u n o p e r a t e d ulcerative colitis. S o m e authors, e m p l o y i n g actuarial m e t h o d s , have calculated a cumulative risk of carc i n o m a after IRA of 13-15 percent, 20-30 years after o n s e t of the disease.3' 4.6 H o w e v e r , the use of actuarial m e t h o d s m a y lead to an o v e r e s t i m a t e of cancer risks. 7 Subjects o p e r a t e d with IRA have l o w e r frequencies of neoplastic transformation w h e n c o m p a r e d with u n o p e r a t e d patients affected by ulcerative colitis. 4 This c o u l d s i m p l y b e the result of a r e d u c t i o n of the m u c o s a at risk in IRA patients, but also of a l o w e r t e n d e n c y of the rectal m u c o s a after IRA to m a l i g n a n t transformation. A correlation b e t w e e n the d e v e l o p m e n t of c o l o n cancer and i n c r e a s e d proliferation a n d / o r disorders in the n o r m a l proliferation pattern a l o n g the crypt has b e e n clearly e s t a b l i s h e d in the past. 8'9 E n h a n c e d cell proliferation and a shift of the proliferative z o n e toward the surface of the crypt has b e e n r e p o r t e d in patients with familial p o l y p o s i s or colorectal cancer and in patients with u n o p e r ated ulcerative colitis, b o t h in active p h a s e or in remission. 1~ O n the contrary, low risk populations for the d e v e l o p m e n t of c o l o n cancer have a q u i e s c e n t proliferative activity in colonic mucosa. 14 O r n i t h i n e d e c a r b o x y l a s e ( O D e ) activity or poly a m i n e tissue levels have also b e e n u s e d as a m e a s u r e of tissue proliferative activity. 15 Polyam i n e s (putrescine, s p e r m i d i n e , and s p e r m i n e ) facilitate transcription, translation, initiation of protein synthesis, and stabilization of p o l y n u c l e o tides. ~6 O r n i t h i n e d e c a r b o x y l a s e is the k e y e n z y m e in the synthesis of p o l y a m i n e s and catalyzes the
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transformation of ornithine into putrescine. 15 ODC activity is correlated with polyamine levels and is varied in premalignant and malignant conditions in humans. 17q9 Elevated ODC activity was found in familial colon polyposis concomitantly with high levels of polyamines. 2~ A previous investigation, examining the epithelial cell proliferation of the rectal stump in patients after IRA for ulcerative colitis, showed that the labeling index was still higher than normal. 21 Because these results were contradictory with the lower cancer risk calculated for IRA patients, we thought it of interest to further examine the proliferative pattern and mucosal polyamine levels in the rectal epithelium in unoperated ulcerative colitis patients and in ulcerative colitis patients after total colectomy and IRA. PATIENTS AND M E T H O D S Because our main goal was to study the effects of colectomy and IRA on the proliferation rate of ulcerative colitis patients, we tried to match the two populations in terms of age, sex, duration of disease, and severity of proctitis, as explained in what follows. Three groups of patients were studied. Group I was composed of 10 patients (5 males, 5 females, mean age 43, range 17-60 years) who had undergone IRA for ulcerative colitis. The mean duration of the disease was 6.6 years (range 2-14); the mean period after surgery was 3 years (range 6 months to 4 years). In all subjects the disease was extended throughout the colon. The severity of proctitis was determined according to Baron's classification22 and was distributed as follows: Grade 0 : 4 cases; Grade 1:5 cases; Grade 2 : 1 case. No cancer or dysplasia were observed on surgical specimens. Four patients were taking 5 aminosalicylic acid (4 g/day) and one methylprednisolone (20 mg/day) by enema. Group II was composed of 10 patients (5 males, 5 females, mean age 43, range 13-62 years) with chronic ulcerative colitis. The mean duration of the disease was 5.7 years (range 2-13). Proctitis was distributed as follows: Grade 0:3 cases; Grade 1:6 cases; Grade 2:1 case. Six cases had diffuse colitis and four cases left-side colitis. Four patients were taking 5 aminosalicylic acid (4 g/day) by enema and six were taking salicylazosulfapyridine (2 g/ day) orally; two patients were also taking corticosteroids (8-20 mg/day) by enema.
Dis Colon Rectum, May 1991
Group III had seven subjects (5 males, 2 females, mean age 48.7, range 32-67 years), showing endoscopically and histologically normal rectal mucosa without any digestive tract disease and no family history of cancer, who were examined with colonoscopy and underwent rectal biopsies for diagnostic purposes, were selected as controls. The mean age of the control group was slightly higher, and the range was displaced toward older ages, given the difficulties of obtaining biopsies from young subjects. Informed consent was obtained from all subjects. Three biopsies of about 1.5 mm 2 (approximate weight 3-10 mg) were taken from each patient at 10 cm from the anal verge by means of an optic fiber colonoscope and transferred in a plastic vial containing 10 ml of o~-MEM (Flow Laboratories, UK). 3H-thymidine incorporation and polyamine levels were then determined. In
Vitro 3H-Thymidine Incorporation and
Autoradiographic Procedures 3H-thymidine uptake was determined in two biopsies with the method described by Stadler et al. 23 with minor modifications. The biopsy specimens were examined and cleaned under a dissecting microscope and then transferred to a 20-ml sterile vial fitted with a sleeve style rubber stopper (Wheaton, MD) and containing 1 ml of ~-MEM medium; 5 ml of 3H-thymidine (Amersham, UK, specific activity 41 Ci/mmol) were added to give a final concentration of 5 vCi/ml, and 25 ml of oxygen were injected in each vial. The vials were then incubated at 37~ for 90 minutes with shaking in a Dubnoff incubator. At the end of the incubation, the pressure was released with a syringe and the specimens washed 10 times in saline at room temperature. The biopsies were then oriented (mucosa side up) under a dissecting microscope and set in a plastic frame larger than the biopsy. A 2 percent agar solution at 40~ was then poured over the biopsy to fix it in the right orientation. Immediately after solidification of the agar solution, the biopsies were fixed in 10 percent buffered formalin. The specimens were then dehydrated and embedded in paraffin; from each biopsy three sections (separated by more than 200 ~m), 5 ~m in thickness and perpendicular to the surface of the mucosa, were obtained. Slides were dipped in the dark in Nuclear Track Emulsion (Kodak NTB-2) diluted 50 percent in distilled water at 40~ and kept in the dark at
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MUCOSAL CELL PROLIFERATION OF RECTAL STUMP
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4 ~C for 2-3 weeks. Slides were then developed by standard photographic procedures and stained with hematoxylin-eosin. Full longitudinal crypt sections were analyzed under a microscope with codes unknown to the operator; an average of 12 crypts/ patients were scored. For each crypt we recorded: number of labeled ceils, position of labeled cells along the crypt, and number of cells scored. Data on cell proliferation were expressed as labeling index (number of labeled cells/number of cells scored Xl00). To evaluate the distribution of labeled cells along the crypt, each crypt was divided into three equal sectors, Sector 1 being the bottom and Sector 3 the top of the crypt. Labeling index (number of labeled ceils in the sector/number of cells counted in that sector x 100) and the percentage of labeled cells (number of labeled ceils in the sector/number of labeled ceils in that crypt x 100) were calculated for each sector of the crypt.
An aliquot of 20 #1 was used for injection in a liquid-liquid chromatograph. A 5-/,m inverse phase Perkin-Elmer C 8 column (0.46 cm ID, 25 cm length) was used with a PerkinElmer fluorescence detector with a 3-#1 flow cell (excitation wavelength 345 nm and emission 518 nm). The detector was equipped with a Hitachi integrator. The eluting solution was a linear CH3 OH:H20 gradient, varying from 60 to 90 percent methanol in 20 minutes. The flow was adjusted to 1.4 ml/min at room temperature. Three polyamines (putrescine, spermidine, and spermine) were identified by comparing the retention times with those of standards (Sigma) and quantified by comparing the integrated peak areas with those of the internal standard (1,8-diaminoctane). Protein content of the biopsies was determined in the homogenate according to Bradford, 24 using bovine serum albumin as a standard. Results were expressed as #g of polyamines/mg protein content.
Determination of Polyamines
Statistical Analysis
We determined the levels of polyamines (putrescine, spermidine, and spermine) in the biopsies with the following method. One biopsy was kept at - 8 0 ~ for a period not exceeding 30 days. Preliminary controls had shown that polyamines levels are stable within this period. Biopsies were weighed and homogenized (1:200; mg tissue: #1 solution) in 0.01 M phosphate buffer/EDTA pH 7.4 containing 1 percent monothioglycerol (Sigma, St. Louis, MO) at 0~ using a glass microhomogenizer. The homogenates were centrifuged at 4,500 x g for 20 minutes. Each supernatant (200 #1) was added with 50/~1 of HC104 1 N and 1,8-diaminoctane as internal standard (2 #g/ml); the mixture was left standing for 5 minutes at room temperature. After centrifugation at 4,500 x g for 10 minutes, an aliquot of 100 #1 of the supernatant was treated to obtain dansyl derivatives with the following procedure: 100/~1 of the supernatant, 100 #1 of 4 N NaHCO3, and 300 ~,1 of dansyl chloride (5 mg/ ml in acetone) were incubated for 18 hours at room temperature in the dark. After the incubation, 20 #1 of proline (1 mg/ml) were added; the solution was left standing for 30 minutes, and dried with a nitrogen flux. The residue was dissolved in 300 #1 of bidistilled water and extracted twice with 5 ml of toluene for 30 seconds. An aliquot of 4 ml of the toluene phase was dried with nitrogen, and the residue dissolved in 150 #1 of H20:CH3OH (50:50).
Data were analyzed with one way analysis of variance using the Statgraphic Statistical Package. RESULTS The labeling index was significantly increased in ulcerative colitis patients (UC) in respect to controls (Fig. 1). IRA patients and controls had similar values of proliferation. The pattern of proliferation along the crypt is reported as the labeling index in different sectors of the crypt and as the percentage of labeled cells in three sectors. The labeling index was higher for UC patients in all compartments of the crypt (Fig. 2). IRA patients did not differ significantly from controls and the labeling index was lower in all three sectors when compared with UC patients. In controls, cell proliferation is located mainly in the lower compartment of the crypt (80 percent of labeled cells are in this zone) (Fig. 3). In UC patients, the pattern of proliferation is significantly shifted toward the transitional and mature zones (middle and upper regions, respectively). IRA patients have intermediate values. Figure 4 shows the variation of total and individual polyamines. Total polyamine levels were significantly increased in UC and IRA patients compared to controls (Panel a); putrescine and spermidine were significantly higher in UC than in
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TONELLI E T AL
Dis Colon Rectum, May 1991 DISCUSSION
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Fig. 1. Labeling index in the rectal mucosa of controls, IRA patients, and UC patients. Data are expressed as mean _+ SE. The number of patients is reported over the bars. *P < 0.05 compared with controls.
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It is well k n o w n that the risk of c o l o n cancer in patients with ulcerative colitis increases with the extent and duration of the disease. 25 Previous studies have shown increased epithelial cell proliferation in patients with ulcerative colitis, a shortening of the turnover time in the crypt e p i t h e l i u m of the rectum, and migration of the proliferative zone toward the u p p e r part of the crypts. 12' 13 The alteration of cell renewal was o b s e r v e d both in active ulcerative colitis and during remissions, 12 and was not correlated to the extension of the disease. In our study we e x a m i n e d u n o p e r a t e d ulcerative colitis patients and ulcerative colitis patients after total c o l e c t o m y and IRA. The duration and severity of the disease w e r e similar in the IRA and in the UC group, as e x p l a i n e d in Patients and Methods. Medical treatment was not h o m o g e n e o u s in the two groups; in fact, as corticosteroids and salicylazosulfapyridine are s u p p o s e d to r e d u c e inflammation and the severity of clinical symptoms, m o r e patients w e r e o n therapy in the UC group w h e n c o m p a r e d with the IRA group. However, because mucosal proliferation is not specifically affected by steroids and salicylazosulfapyridine, or at most it might be d e c r e a s e d 12, the different p e r c e n t a g e of patients on drug therapy s h o u l d not have an effect on the decrease of proliferation observed after IRA. The group of patients with IRA was not comp o s e d of selected subjects, since IRA was perf o r m e d by us in the majority of patients (82 percent). However, before surgery, they w e r e in an
100!
6 4 2 0
~D C
IRA
lower
UC
C
IRA
middle
UC
C
IRA
g0 8O 70 50
UC
upper
Fig. 2. Distribution of labeling index along the crypt of rectal mucosa of controls, IRA patients, and UC patients. Data are expressed as mean _+ SE. *P < 0.05 compared with controls.
40 30 20 I0 0
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control mucosa whereas they were n o r m a l i z e d in IRA patients (Panels b and c); s p e r m i n e levels were increased in UC and IRA patients (panel d) w h e n c o m p a r e d with controls.
UC
C
IRA middle
UC
C
IRA
UC
upper
Fig. 3. Distribution of labeled cells along the crypt of rectal mucosa of controls, IRA patients, and UO patients. Data are expressed as mean __+SE. "P < O.05 compared with controls.
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MUCOSAL CELL PROLIFERATION OF RECTAL STUMP
c
g
o
g c
r._ o_
IRA
C
UC C
IRA
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UU
2.5 c ~
2.0
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?_ c:l ~, ~.5
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Fig. 4. Level of total polyamines (Panel b), spermidine (Panel c), and the rectal mucosa of controls, IRA tients. Data are expressed as mean patients is reported over the bars. with controls.
(Panel a), putrescine spermine (Panel d) in patients, and UC pa_+ SE. The number of *P < 0.05 compared
active phase of the disease, refractory to medical treatment. Lehy e t a L 2~ had demonstrated before that the labeling index did not revert to normality in IRA patients. In their study the proliferative compartment was extended in the upper portion of the crypt in UC patients; and this p h e n o m e n o n was decreased, but did not disappear, in patients with IRA. Our results disagree with those reported by Lehy e t aL 21 and show that the labeling index was in the range of controls in patients after IRA. The distribution pattern of the labeling index and the percentage of labeled cells show that UC patients had a displacement of the proliferation toward the upper zones of the crypt, whereas IRA patients did n o t significantly differ from controls. The data also show that total polyamines, spermidine, and spermine were increased in UC patients and this is
389
coherent with a status of increased cellular proliferation. After IRA, the total polyamine content and spermine slightly decreased, although they remained higher than controls, but spermidine and putrescine reverted to normal values. It has been suggested that these three factors (increased colon proliferative activity, a shift of the proliferation from the lower to the upper sections of the crypt, and high levels of polyamines) are associated with an increased risk of developing cancer; for this reason the reported results may explain the increased risk of carcinoma in ulcerative colitis. On the contrary, because IRA patients had a pattern of proliferation similar to controls, our results suggest that IRA patients may have a reduction of rectal cancer risk. This might not be just the result of a reduction of the mucosa at risk for carcinoma, because the rectal ampulla still is the most frequent site of carcinoma in ulcerative colitis. 26 However, the fact that total polyamines and spermine levels were still high after IRA suggest that some biochemical events connected to cell proliferation are not totally normalized by surgery. Cancer cases after IRA usually have severe dysplasia in the excised colon and develop cancer a relatively short period after IRA. 1 It is possible that these patients had severe dysplasia of the rectal mucosa at the m o m e n t of surgery and the develo p m e n t of carcinoma in these patients could not be avoided by late changes in proliferation consequent to total colectomy. We have followed 66 IRA-UC patients with no cancer o r dysplasia at surgery, and no cancer developed after a mean follow-up period of 11.8 years (range 1-26 years) of the disease. 27 Therefore, if we exclude patients in whom IRA was performed in the presence of colon cancer or severe dysplasia at the time of colectomy, this surgical procedure may reduce the incidence of cancer in the rectal stump. We did not carry out a historical study in patients before and after surgery, and this could raise questions about the validity of our conclusions. However, it is reasonable to suppose that the effects shown are not sampling errors, because we matched UC and IRA patients for age, duration of disease, and severity of proctitis. Moreover, the population means are very far apart and the standard error relatively small. We have no information on the mechanism responsible for the reduced proliferative activity of
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the rectal m u c o s a after IRA. R e d u c e d proliferation c o u l d be due to a m o d i f i e d intestinal e n v i r o n m e n t i n d u c e d by surgery ( e . g . , alterations of the qualitative and quantitative pattern of fecal bile acids after surgery), but s o m e other m e c h a n i s m , w h i c h deserves further investigation, might be responsible for the observed effects.
REFERENCES 1. Oakley JR, Jagelman DG, Fazio VW, e t aL Complications and quality of life after ileorectal anastomosis for ulcerative colitis. Am J Surg 1985;149:23-30. 2. Jones PF, Munro A, Ewen SW. Colectomy and ileorectal anastomosis for colitis: report on a personal series, with a critical review. Br J Surg 1977;64:61523. 3. Baker WN, Glass RE, Ritchie JK, Aylett SO. Cancer of the rectum following colectomy and ileorectal anastomosis for ulcerative colitis. Br J Surg 1978;65:862-8. 4. Grundfest SF, Fazio V, Weiss RA, et al. The risk of cancer following colectomy and ileorectal anastomosis for extensive mucosal ulcerative colitis. Ann Surg 1981;193:9-14. 5. Khubchandani IT, Trimpi HD, Sheets JA. Ileorectal anastomosis for ulcerative and Crohn's colitis. Am J Surg 1978;135:751-6. 6. Berard PH, Parc R. Le traitment de la rectocolite ulcerohemorragique. 86 Congres Francais de Chirurgie, Masson, Paris, 1984. 7. YardleyJH, Keren DF. "Precancer" lesions in ulcerative colitis: a retrospective study of rectal biopsy and colectomy specimens. Cancer 1974;34:835-44. 8. Deschner EE, Maskens AP. Significance of the labeling index and labeling distribution as kinetic parameters in colorectal mucosa of cancer patients and DMH treated animals. Cancer 1982;50:1136-41. 9. Lipkin M. Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects. Cancer Res 1988;48:235-45. 10. Deschner EE, Lipkin M. Proliferative patterns in coIonic mucosa in familial polyposis. Cancer 1975;35:413-8. 11. Ponz de Leon M, Roncucci L, Di Donato P, et al. Pattern of epithelial cell proliferation in colorectal mucosa of normal subjects and of patients with adenomatous polyps or cancer of the large bowel. Cancer Res 1988;48:4121-6. 12. Serafini EP, Kirk AP, Chambers TJ. Rate and pattern of epithelial cell proliferation in ulcerative colitis.
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Gut 1981;22:648-52. 13. Bleiberg H, Mainguet P, Galand P, Chretien J, Dupont-Mairesse N. Cell renewal in the human rectum: in vitro autoradiographic study on active ulcerative colitis. Gastroenterology 1970;58:851-5. 14.. Lipkin M, Uehara K, Winawer S, e t al. Seventh-Day Adventist vegetarians have a quiescent proliferative activity in colonic mucosa. Cancer Lett 1985;26:13944. 15. Pegg AE, McCann PP. Polyamine metabolism and functions. Am J Physiol 1982;243:C212-21. 16. Bloomfield VA, Wilson RW. Interactions of polyamines with polynucleotides. In: Morris DR, Marton LJ, eds. Polyamines in biology and medicine. New York: Marcel Dekker, 1981:183-206. 17. LaMuraglia GM, Lacaine F, Malt RA. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann Surg 1986;204:89-93. 18. Garewal HS, Gerner EW, Sampliner RE, Roe D. Omithine decarboxylase and polyamine levels in columnar upper gastrointestinal mucosae in patients with Barrett's esophagus. Cancer Res 1988;48:328891. 19. Porter CW, Herrera-Ornelas L, Pera P, Petrelli NF, Mittelman A. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 1987;60:1275-81. 20. Luk GD, Baylin SB. ODC as a biological marker in familial colonic polyposis. N Engl J Med 1984;311:80-3. 21. Lehy T, Mignon M, Abitbol JL. Epithelial cell proliferation in the rectal stump of patients with ileorectal anastomosis for ulcerative colitis. Gut 1983;24:104856. 22. Baron JH, Connel AM, Lennard-Jones JE. Variation between observers in describing mucosal appearances in proctocolitis. Br Med J 1964;1:89-92. 23. Stadler J, Stern HS, Yeung KS, e t al. Effect of high fat consumption on cell proliferation activity of colorectal mucosa and on soluble faecal bile acids. Gut 1988;29:1326-31. 24. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;72:248-54. 25. Nugent WF, Haggit RC, Colcher H, Kutteruf GC. Malignant potential of chronic ulcerative colitis. Gastroenterology 1979;76:1-5. 26. Gyde SN, Prior P, Thompson H. Survival of patients with colorectal cancer complicating ulcerative colitis. Gut 1984;25:228-31. 27. Tonelli F. Terapia chirurgica della colite ulcerosa: i risultati dell'ileo-retto anastomosi. 88 ~ Congresso Soc Ital Chir, Pozzi, Roma, 1986.
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atients affected b y ulcerative colitis requiring c o l e c t o m y can p r e s e r v e c o n t i n e n c e and avoid a stoma b y two surgical p r o c e d u r e s : the ileorectal a n a s t o m o s i s (IRA) or the ileoanal anastomosis. T h e f o r m e r is a s i m p l e r t e c h n i q u e and, if p e r f o r m e d on p r o p e r l y s e l e c t e d patients, g u a r a n t e e s satisfactory functional results. ~' 2 H o w e v e r , leaving the d i s e a s e d r e c t u m in place m a y e x p o s e the patient to the risk
This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro, from CNR "Progetto Finalizzato FATMA"and by MPI, Italy. Address reprint requests to Professor Tonelli: Department of Clinical Physiopathology, Viale Morgagni 85, 50134 Florence, Italy. 385
of rectal carcinoma. This c o m p l i c a t i o n s e e m s to b e rare (varying f r o m 0 to 6 p e r c e n t in all patients3-5), but a reliable m e a s u r e of the risk is difficult to obtain b e c a u s e the risk of d e v e l o p i n g c a r c i n o m a increases with the duration of the disease, similar to what h a p p e n s in patients with u n o p e r a t e d ulcerative colitis. S o m e authors, e m p l o y i n g actuarial m e t h o d s , have calculated a cumulative risk of carc i n o m a after IRA of 13-15 percent, 20-30 years after o n s e t of the disease.3' 4.6 H o w e v e r , the use of actuarial m e t h o d s m a y lead to an o v e r e s t i m a t e of cancer risks. 7 Subjects o p e r a t e d with IRA have l o w e r frequencies of neoplastic transformation w h e n c o m p a r e d with u n o p e r a t e d patients affected by ulcerative colitis. 4 This c o u l d s i m p l y b e the result of a r e d u c t i o n of the m u c o s a at risk in IRA patients, but also of a l o w e r t e n d e n c y of the rectal m u c o s a after IRA to m a l i g n a n t transformation. A correlation b e t w e e n the d e v e l o p m e n t of c o l o n cancer and i n c r e a s e d proliferation a n d / o r disorders in the n o r m a l proliferation pattern a l o n g the crypt has b e e n clearly e s t a b l i s h e d in the past. 8'9 E n h a n c e d cell proliferation and a shift of the proliferative z o n e toward the surface of the crypt has b e e n r e p o r t e d in patients with familial p o l y p o s i s or colorectal cancer and in patients with u n o p e r ated ulcerative colitis, b o t h in active p h a s e or in remission. 1~ O n the contrary, low risk populations for the d e v e l o p m e n t of c o l o n cancer have a q u i e s c e n t proliferative activity in colonic mucosa. 14 O r n i t h i n e d e c a r b o x y l a s e ( O D e ) activity or poly a m i n e tissue levels have also b e e n u s e d as a m e a s u r e of tissue proliferative activity. 15 Polyam i n e s (putrescine, s p e r m i d i n e , and s p e r m i n e ) facilitate transcription, translation, initiation of protein synthesis, and stabilization of p o l y n u c l e o tides. ~6 O r n i t h i n e d e c a r b o x y l a s e is the k e y e n z y m e in the synthesis of p o l y a m i n e s and catalyzes the
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transformation of ornithine into putrescine. 15 ODC activity is correlated with polyamine levels and is varied in premalignant and malignant conditions in humans. 17q9 Elevated ODC activity was found in familial colon polyposis concomitantly with high levels of polyamines. 2~ A previous investigation, examining the epithelial cell proliferation of the rectal stump in patients after IRA for ulcerative colitis, showed that the labeling index was still higher than normal. 21 Because these results were contradictory with the lower cancer risk calculated for IRA patients, we thought it of interest to further examine the proliferative pattern and mucosal polyamine levels in the rectal epithelium in unoperated ulcerative colitis patients and in ulcerative colitis patients after total colectomy and IRA. PATIENTS AND M E T H O D S Because our main goal was to study the effects of colectomy and IRA on the proliferation rate of ulcerative colitis patients, we tried to match the two populations in terms of age, sex, duration of disease, and severity of proctitis, as explained in what follows. Three groups of patients were studied. Group I was composed of 10 patients (5 males, 5 females, mean age 43, range 17-60 years) who had undergone IRA for ulcerative colitis. The mean duration of the disease was 6.6 years (range 2-14); the mean period after surgery was 3 years (range 6 months to 4 years). In all subjects the disease was extended throughout the colon. The severity of proctitis was determined according to Baron's classification22 and was distributed as follows: Grade 0 : 4 cases; Grade 1:5 cases; Grade 2 : 1 case. No cancer or dysplasia were observed on surgical specimens. Four patients were taking 5 aminosalicylic acid (4 g/day) and one methylprednisolone (20 mg/day) by enema. Group II was composed of 10 patients (5 males, 5 females, mean age 43, range 13-62 years) with chronic ulcerative colitis. The mean duration of the disease was 5.7 years (range 2-13). Proctitis was distributed as follows: Grade 0:3 cases; Grade 1:6 cases; Grade 2:1 case. Six cases had diffuse colitis and four cases left-side colitis. Four patients were taking 5 aminosalicylic acid (4 g/day) by enema and six were taking salicylazosulfapyridine (2 g/ day) orally; two patients were also taking corticosteroids (8-20 mg/day) by enema.
Dis Colon Rectum, May 1991
Group III had seven subjects (5 males, 2 females, mean age 48.7, range 32-67 years), showing endoscopically and histologically normal rectal mucosa without any digestive tract disease and no family history of cancer, who were examined with colonoscopy and underwent rectal biopsies for diagnostic purposes, were selected as controls. The mean age of the control group was slightly higher, and the range was displaced toward older ages, given the difficulties of obtaining biopsies from young subjects. Informed consent was obtained from all subjects. Three biopsies of about 1.5 mm 2 (approximate weight 3-10 mg) were taken from each patient at 10 cm from the anal verge by means of an optic fiber colonoscope and transferred in a plastic vial containing 10 ml of o~-MEM (Flow Laboratories, UK). 3H-thymidine incorporation and polyamine levels were then determined. In
Vitro 3H-Thymidine Incorporation and
Autoradiographic Procedures 3H-thymidine uptake was determined in two biopsies with the method described by Stadler et al. 23 with minor modifications. The biopsy specimens were examined and cleaned under a dissecting microscope and then transferred to a 20-ml sterile vial fitted with a sleeve style rubber stopper (Wheaton, MD) and containing 1 ml of ~-MEM medium; 5 ml of 3H-thymidine (Amersham, UK, specific activity 41 Ci/mmol) were added to give a final concentration of 5 vCi/ml, and 25 ml of oxygen were injected in each vial. The vials were then incubated at 37~ for 90 minutes with shaking in a Dubnoff incubator. At the end of the incubation, the pressure was released with a syringe and the specimens washed 10 times in saline at room temperature. The biopsies were then oriented (mucosa side up) under a dissecting microscope and set in a plastic frame larger than the biopsy. A 2 percent agar solution at 40~ was then poured over the biopsy to fix it in the right orientation. Immediately after solidification of the agar solution, the biopsies were fixed in 10 percent buffered formalin. The specimens were then dehydrated and embedded in paraffin; from each biopsy three sections (separated by more than 200 ~m), 5 ~m in thickness and perpendicular to the surface of the mucosa, were obtained. Slides were dipped in the dark in Nuclear Track Emulsion (Kodak NTB-2) diluted 50 percent in distilled water at 40~ and kept in the dark at
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MUCOSAL CELL PROLIFERATION OF RECTAL STUMP
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4 ~C for 2-3 weeks. Slides were then developed by standard photographic procedures and stained with hematoxylin-eosin. Full longitudinal crypt sections were analyzed under a microscope with codes unknown to the operator; an average of 12 crypts/ patients were scored. For each crypt we recorded: number of labeled ceils, position of labeled cells along the crypt, and number of cells scored. Data on cell proliferation were expressed as labeling index (number of labeled cells/number of cells scored Xl00). To evaluate the distribution of labeled cells along the crypt, each crypt was divided into three equal sectors, Sector 1 being the bottom and Sector 3 the top of the crypt. Labeling index (number of labeled ceils in the sector/number of cells counted in that sector x 100) and the percentage of labeled cells (number of labeled ceils in the sector/number of labeled ceils in that crypt x 100) were calculated for each sector of the crypt.
An aliquot of 20 #1 was used for injection in a liquid-liquid chromatograph. A 5-/,m inverse phase Perkin-Elmer C 8 column (0.46 cm ID, 25 cm length) was used with a PerkinElmer fluorescence detector with a 3-#1 flow cell (excitation wavelength 345 nm and emission 518 nm). The detector was equipped with a Hitachi integrator. The eluting solution was a linear CH3 OH:H20 gradient, varying from 60 to 90 percent methanol in 20 minutes. The flow was adjusted to 1.4 ml/min at room temperature. Three polyamines (putrescine, spermidine, and spermine) were identified by comparing the retention times with those of standards (Sigma) and quantified by comparing the integrated peak areas with those of the internal standard (1,8-diaminoctane). Protein content of the biopsies was determined in the homogenate according to Bradford, 24 using bovine serum albumin as a standard. Results were expressed as #g of polyamines/mg protein content.
Determination of Polyamines
Statistical Analysis
We determined the levels of polyamines (putrescine, spermidine, and spermine) in the biopsies with the following method. One biopsy was kept at - 8 0 ~ for a period not exceeding 30 days. Preliminary controls had shown that polyamines levels are stable within this period. Biopsies were weighed and homogenized (1:200; mg tissue: #1 solution) in 0.01 M phosphate buffer/EDTA pH 7.4 containing 1 percent monothioglycerol (Sigma, St. Louis, MO) at 0~ using a glass microhomogenizer. The homogenates were centrifuged at 4,500 x g for 20 minutes. Each supernatant (200 #1) was added with 50/~1 of HC104 1 N and 1,8-diaminoctane as internal standard (2 #g/ml); the mixture was left standing for 5 minutes at room temperature. After centrifugation at 4,500 x g for 10 minutes, an aliquot of 100 #1 of the supernatant was treated to obtain dansyl derivatives with the following procedure: 100/~1 of the supernatant, 100 #1 of 4 N NaHCO3, and 300 ~,1 of dansyl chloride (5 mg/ ml in acetone) were incubated for 18 hours at room temperature in the dark. After the incubation, 20 #1 of proline (1 mg/ml) were added; the solution was left standing for 30 minutes, and dried with a nitrogen flux. The residue was dissolved in 300 #1 of bidistilled water and extracted twice with 5 ml of toluene for 30 seconds. An aliquot of 4 ml of the toluene phase was dried with nitrogen, and the residue dissolved in 150 #1 of H20:CH3OH (50:50).
Data were analyzed with one way analysis of variance using the Statgraphic Statistical Package. RESULTS The labeling index was significantly increased in ulcerative colitis patients (UC) in respect to controls (Fig. 1). IRA patients and controls had similar values of proliferation. The pattern of proliferation along the crypt is reported as the labeling index in different sectors of the crypt and as the percentage of labeled cells in three sectors. The labeling index was higher for UC patients in all compartments of the crypt (Fig. 2). IRA patients did not differ significantly from controls and the labeling index was lower in all three sectors when compared with UC patients. In controls, cell proliferation is located mainly in the lower compartment of the crypt (80 percent of labeled cells are in this zone) (Fig. 3). In UC patients, the pattern of proliferation is significantly shifted toward the transitional and mature zones (middle and upper regions, respectively). IRA patients have intermediate values. Figure 4 shows the variation of total and individual polyamines. Total polyamine levels were significantly increased in UC and IRA patients compared to controls (Panel a); putrescine and spermidine were significantly higher in UC than in
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TONELLI E T AL
Dis Colon Rectum, May 1991 DISCUSSION
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,--,
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Fig. 1. Labeling index in the rectal mucosa of controls, IRA patients, and UC patients. Data are expressed as mean _+ SE. The number of patients is reported over the bars. *P < 0.05 compared with controls.
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02
8
It is well k n o w n that the risk of c o l o n cancer in patients with ulcerative colitis increases with the extent and duration of the disease. 25 Previous studies have shown increased epithelial cell proliferation in patients with ulcerative colitis, a shortening of the turnover time in the crypt e p i t h e l i u m of the rectum, and migration of the proliferative zone toward the u p p e r part of the crypts. 12' 13 The alteration of cell renewal was o b s e r v e d both in active ulcerative colitis and during remissions, 12 and was not correlated to the extension of the disease. In our study we e x a m i n e d u n o p e r a t e d ulcerative colitis patients and ulcerative colitis patients after total c o l e c t o m y and IRA. The duration and severity of the disease w e r e similar in the IRA and in the UC group, as e x p l a i n e d in Patients and Methods. Medical treatment was not h o m o g e n e o u s in the two groups; in fact, as corticosteroids and salicylazosulfapyridine are s u p p o s e d to r e d u c e inflammation and the severity of clinical symptoms, m o r e patients w e r e o n therapy in the UC group w h e n c o m p a r e d with the IRA group. However, because mucosal proliferation is not specifically affected by steroids and salicylazosulfapyridine, or at most it might be d e c r e a s e d 12, the different p e r c e n t a g e of patients on drug therapy s h o u l d not have an effect on the decrease of proliferation observed after IRA. The group of patients with IRA was not comp o s e d of selected subjects, since IRA was perf o r m e d by us in the majority of patients (82 percent). However, before surgery, they w e r e in an
100!
6 4 2 0
~D C
IRA
lower
UC
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IRA
middle
UC
C
IRA
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upper
Fig. 2. Distribution of labeling index along the crypt of rectal mucosa of controls, IRA patients, and UC patients. Data are expressed as mean _+ SE. *P < 0.05 compared with controls.
40 30 20 I0 0
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control mucosa whereas they were n o r m a l i z e d in IRA patients (Panels b and c); s p e r m i n e levels were increased in UC and IRA patients (panel d) w h e n c o m p a r e d with controls.
UC
C
IRA middle
UC
C
IRA
UC
upper
Fig. 3. Distribution of labeled cells along the crypt of rectal mucosa of controls, IRA patients, and UO patients. Data are expressed as mean __+SE. "P < O.05 compared with controls.
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MUCOSAL CELL PROLIFERATION OF RECTAL STUMP
c
g
o
g c
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Fig. 4. Level of total polyamines (Panel b), spermidine (Panel c), and the rectal mucosa of controls, IRA tients. Data are expressed as mean patients is reported over the bars. with controls.
(Panel a), putrescine spermine (Panel d) in patients, and UC pa_+ SE. The number of *P < 0.05 compared
active phase of the disease, refractory to medical treatment. Lehy e t a L 2~ had demonstrated before that the labeling index did not revert to normality in IRA patients. In their study the proliferative compartment was extended in the upper portion of the crypt in UC patients; and this p h e n o m e n o n was decreased, but did not disappear, in patients with IRA. Our results disagree with those reported by Lehy e t aL 21 and show that the labeling index was in the range of controls in patients after IRA. The distribution pattern of the labeling index and the percentage of labeled cells show that UC patients had a displacement of the proliferation toward the upper zones of the crypt, whereas IRA patients did n o t significantly differ from controls. The data also show that total polyamines, spermidine, and spermine were increased in UC patients and this is
389
coherent with a status of increased cellular proliferation. After IRA, the total polyamine content and spermine slightly decreased, although they remained higher than controls, but spermidine and putrescine reverted to normal values. It has been suggested that these three factors (increased colon proliferative activity, a shift of the proliferation from the lower to the upper sections of the crypt, and high levels of polyamines) are associated with an increased risk of developing cancer; for this reason the reported results may explain the increased risk of carcinoma in ulcerative colitis. On the contrary, because IRA patients had a pattern of proliferation similar to controls, our results suggest that IRA patients may have a reduction of rectal cancer risk. This might not be just the result of a reduction of the mucosa at risk for carcinoma, because the rectal ampulla still is the most frequent site of carcinoma in ulcerative colitis. 26 However, the fact that total polyamines and spermine levels were still high after IRA suggest that some biochemical events connected to cell proliferation are not totally normalized by surgery. Cancer cases after IRA usually have severe dysplasia in the excised colon and develop cancer a relatively short period after IRA. 1 It is possible that these patients had severe dysplasia of the rectal mucosa at the m o m e n t of surgery and the develo p m e n t of carcinoma in these patients could not be avoided by late changes in proliferation consequent to total colectomy. We have followed 66 IRA-UC patients with no cancer o r dysplasia at surgery, and no cancer developed after a mean follow-up period of 11.8 years (range 1-26 years) of the disease. 27 Therefore, if we exclude patients in whom IRA was performed in the presence of colon cancer or severe dysplasia at the time of colectomy, this surgical procedure may reduce the incidence of cancer in the rectal stump. We did not carry out a historical study in patients before and after surgery, and this could raise questions about the validity of our conclusions. However, it is reasonable to suppose that the effects shown are not sampling errors, because we matched UC and IRA patients for age, duration of disease, and severity of proctitis. Moreover, the population means are very far apart and the standard error relatively small. We have no information on the mechanism responsible for the reduced proliferative activity of
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TONELLI E T AL
the rectal m u c o s a after IRA. R e d u c e d proliferation c o u l d be due to a m o d i f i e d intestinal e n v i r o n m e n t i n d u c e d by surgery ( e . g . , alterations of the qualitative and quantitative pattern of fecal bile acids after surgery), but s o m e other m e c h a n i s m , w h i c h deserves further investigation, might be responsible for the observed effects.
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Gut 1981;22:648-52. 13. Bleiberg H, Mainguet P, Galand P, Chretien J, Dupont-Mairesse N. Cell renewal in the human rectum: in vitro autoradiographic study on active ulcerative colitis. Gastroenterology 1970;58:851-5. 14.. Lipkin M, Uehara K, Winawer S, e t al. Seventh-Day Adventist vegetarians have a quiescent proliferative activity in colonic mucosa. Cancer Lett 1985;26:13944. 15. Pegg AE, McCann PP. Polyamine metabolism and functions. Am J Physiol 1982;243:C212-21. 16. Bloomfield VA, Wilson RW. Interactions of polyamines with polynucleotides. In: Morris DR, Marton LJ, eds. Polyamines in biology and medicine. New York: Marcel Dekker, 1981:183-206. 17. LaMuraglia GM, Lacaine F, Malt RA. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann Surg 1986;204:89-93. 18. Garewal HS, Gerner EW, Sampliner RE, Roe D. Omithine decarboxylase and polyamine levels in columnar upper gastrointestinal mucosae in patients with Barrett's esophagus. Cancer Res 1988;48:328891. 19. Porter CW, Herrera-Ornelas L, Pera P, Petrelli NF, Mittelman A. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 1987;60:1275-81. 20. Luk GD, Baylin SB. ODC as a biological marker in familial colonic polyposis. N Engl J Med 1984;311:80-3. 21. Lehy T, Mignon M, Abitbol JL. Epithelial cell proliferation in the rectal stump of patients with ileorectal anastomosis for ulcerative colitis. Gut 1983;24:104856. 22. Baron JH, Connel AM, Lennard-Jones JE. Variation between observers in describing mucosal appearances in proctocolitis. Br Med J 1964;1:89-92. 23. Stadler J, Stern HS, Yeung KS, e t al. Effect of high fat consumption on cell proliferation activity of colorectal mucosa and on soluble faecal bile acids. Gut 1988;29:1326-31. 24. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;72:248-54. 25. Nugent WF, Haggit RC, Colcher H, Kutteruf GC. Malignant potential of chronic ulcerative colitis. Gastroenterology 1979;76:1-5. 26. Gyde SN, Prior P, Thompson H. Survival of patients with colorectal cancer complicating ulcerative colitis. Gut 1984;25:228-31. 27. Tonelli F. Terapia chirurgica della colite ulcerosa: i risultati dell'ileo-retto anastomosi. 88 ~ Congresso Soc Ital Chir, Pozzi, Roma, 1986.
